Charité- Universitätsmedizin Berlin
Campus Benjamin Franklin
Aus der Klinik für Nephrologie (Medizinische Klinik IV)
Geschäftsführender Direktor: Prof. Dr. Zidek
Abteilung: Bioanalytisches Labor
Abteilungsleiter: Prof. Dr. Schlüter
Purification and characterization of angiotensin-II-generating enzymes from
porcine renal tissue
Inaugural-Dissertation
zur Erlangung des Grades
Doctor rerum medicarum
der Charité- Universitätsmedizin Berlin
Campus Benjamin Franklin
vorgelegt von Jana Rykl
aus Strausberg
Referent: Prof. Dr. rer. nat. H. Schlüter
Korreferent: Prof. Dr. med. Th. Unger
Gedruckt mit Genehmigung der Charité- Universitätsmedizin Berlin
Campus Benjamin Franklin
Summary page 88 Summary
The object of this work was the purification, characterization and identification of angiotensin-II-generating enzymes from porcine renal tissue. Three different angiotensin-II-angiotensin-II-generating enzymes were purified using protein liquid chromatography techniques. During the course of purification angiotensin-II-generating enzyme activity was measured and characterized using a novel mass spectrometry based assay system (MES-assay system) for the detection of proteolytic enzyme activities.
One active fraction, here named Fraction I was purified using a combination of anion exchange, hydroxyapatite, lectin affinity and chymostatin-antipain-inhibitor affinity chromatography. Protein identification experiments using peptide mass fingerprint identified this enzyme as Cathepsin G. Cathepsin G was detected in kidney for the first time. The second active fraction, here named Fraction II was purified using a combination of anion exchange, hydroxyapatite, and lentil lectin sugar affinity and size exclusion chromatography. Protein identification experiments identified this enzyme as Angiotensin converting enzyme. The successful purification of ACE is a proof of principle for the chosen purification strategy and the novel MES-assay system.
The third angiotensin-II-generating enzyme fraction, here named Fraction III was purified using a combination of anion exchange, wheat germ lectin sugar affinity and aprotinin-inhibitor affinity chromatography. The identification of this enzyme did not succeed. Experiments in order to characterize this enzyme fraction were carried out. The molecular weight of the enzyme fraction was approximately 160 kDa. A unique feature of Fraction III was its substrate specifity. Besides its angiotensin-II-generating activity it cleaved the L-kininogen analogue Lys-Bradykinin-Ser-Val-Gln-Val-Ser to form Des-Arg10-Kallidin. The proteolytic activity of Fraction III does not coincide with the known proteases of the Kinintensin system. Further research efforts should be made to reveal the identity of this enzyme.
Zusammenfassung page 89 Zusammenfassung
Das Thema dieser Arbeit war die Reinigung, Charakterisierung und Identifizierung von Angiotensin- II generierenden Enzymen aus der Schweineniere. Drei verschiedene Angiotensin- II generierende Enzyme wurden mit Hilfe der Flüssigchromatographie aufgereinigt. Die Angiotensin-II generierende Enzymaktiviät wurde während der Aufreinigung mittels eines neuen Massenspekrometrie basierten Nachweissystems (MES-System) detektiert und charakterisiert.
Eine aktive Fraktion, hier Fraktion I genannt, wurde mit einer Kombination von Anionenaustausch-, Hydroxyapatit-, Lectin- und Chymostatin-Antipain-Affinitätschromatographie gereinigt. Mit Hilfe von Proteinidentifizierungsexperimenten wurde das Angiotensin-II generierende Enzym dieser Fraktion als Cathepsin G identifiziert. Cathepsin G wurde das erste Mal in der Niere nachgewiesen. Eine zweite aktive Fraktion, hier Fraktion II genannt, wurde mit einer Kombination von Anionenaustausch-, Hydroxyapatit-, Lectin- und Größenausschlusschromatographie gereinigt. Mit Hilfe von Proteinidenfizierungsexperimenten wurde das Angiotensin-II generierende Enzym dieser Fraktion als Angiotensin Converting Enzym identifiziert. Die erfolgreiche Aufreinigung ein Beweis für das Funktionieren der gewählten Aufreinigungsstrategie und des neuartigen MES- Nachweissystems. Eine dritte aktive Fraktion, hier Fraktion III genannt, wurde mittel einer Kombination aus Anionenaustausch-, Lectin- und Aprotinin- Affinitätschromatographie aufgereinigt. Die Identifizierung des Angiotensin-II generierenden Enzyms dieser Fraktion gelang nicht. Experimente zur Charakterisierung dieser Enzymfraktion wurden durchgeführt. Das Molekulargewicht betrug ca. 160 kDa. Ein besonderes Merkmal dieser Enzymfraktion war die Subtratspezifität. Neben der Angiotensin-II generierende Aktivität spaltete es das L-Kininogen Analogon Lys-Bradikin-Ser-Val-Gln-Ser Substrat proteolytisch zu Des-Arg10-Kallidin. Weitere Anstrengungen müssen unternommen werden, um die Identität dieses Enzyms zu klären.
Table of content
1. Introduction p.5
2. The aim of the study p.11
3. Methods p.12
3.1 General part p.12
3.1.1 Matrix-assisted-laser-desorption-ionization-time- of- flight-mass-spectrometry
p.12
3.1.2 MALDI-TOF- mass-spectrometry-assisted enzyme screening (MES) system p.13
3.1.3 Chromatography p.14
3.1.3.1 Sample displacement chromatography p.14 3.1.3.2 The protein- purification- parameter-screening system (PPS) p.15
3.1.3.3 Ion exchange chromatography p.16
3.1.3.4 Hydrophobic interaction chromatography (HIC) p.17
3.1.3.5 Hydroxyapatite chromatography p.17
3.1.3.6 Affinity chromatography of glycoproteins p.17 3.1.3.7 Inhibitor affinity chromatography of enzymes p.18
3.1.3.8 Size exclusion chromatography p.18
3.1.4 One- dimensional gel electrophoresis p.19 3.1.5 Two-dimensional gel electrophoresis p.20 3.1.6 Identification of proteins by MALDI-TOF peptide mass fingerprint p.21 3.1.7 Identification of proteins by nano Liquid-Chromatography (nanoLC)
coupled with Electrospray-Ionization tandem mass spectrometry (ESI-MS/MS)
p.22
3.2 Purification of Angiotensin-II-generating enzymes p.23 3.2.1 Part I- Detection of enzymatic products of the renin-angiotensin-system
(RAS) with the MES- Assay
p.27
3.2.1.1 Tissue homogenization and preparation of the protein extract p.27 3.2.1.2 Detection of angiotensin-II-generating activities of the pure porcine
Angiotensin converting enzyme (ACE) and porcine renal tissue protein extract with the MES-assay
p.27
3.2.1.3 Correlation between enzyme activity and MES-signal intensity p.29 3.2.1.4 Comparison of the detection sensitivity of ACE between MES- and
fluorometric enzyme assay
3.2.1.5 Measurement of angiotensin-II-generating activity of renal tissue in presence of protease inhibitors
p.30
3.2.2 Part II-Purification of two angiotensin-II-generating enzymes from porcine renal tissue protein extract
p.31
3.2.2.1 Protein purification scouting (PPS) experiments for the determination of parameters for anion exchange chromatography
p.31
3.2.2.2 Anion exchange chromatography of porcine renal tissue protein extract p.32 3.2.2.3 Inhibitor profiling of the eluates with angiotensin-II- generating activities p.33 3.2.2.4 Protein purification scouting (PPS) experiments for the determination of
parameters of the intermediate chromatographic purification step
p.33
3.2.2.5 Hydroxyapatite chromatography p.35
3.2.2.6 Protein purification scouting (PPS) experiments for the determination of parameters of sugar affinity chromatography
p.35
3.2.2.7 Lectin affinity chromatography p.36
3.2.2.8 Inhibitor profiling of the eluates from lectin affinity chromatography with angiotensin-II- generating activities
p.37
3.2.2.9 Chymostatin-Antipain affinity chromatography p.37
3.2.2.10 Size exclusion chromatography p.38
3.2.3 Part III- Purification of an angiotensin-II-generating fraction from porcine renal tissue protein extract
p.39
3.2.3.1 Anion exchange chromatography of porcine renal tissue protein extract p.39
3.2.3.2 Wheat Germ Lectin chromatography p.40
3.2.3.3 Inhibitor profiling of the eluates with angiotensin-II- generating activities p.40
3.2.3.4 Aprotinin affinity chromatography p.41
3.2.3.5 Determination of the molecular weight of Fraction III p.41 3.2.3.6 Test on substrate specifity of Fraction III p.42 3.2.3.7 Database matching of the retrieved substrate specifity information of
Fraction III
p.42
3.2.4 PART IV- Protein identification of the Fractions I, II, and III p.43 3.2.4.1 Separation of proteins by gel electrophoresis p.43 3.2.4.2 In- gel digestion of protein bands with specific endopeptidases p.43
3.2.4.3 Peptide mass fingerprint p.44
4. Results p.46 4.1 Part I Detection of enzymatic products of the renin-angiotensin-system
(RAS) with the MES- Assay
p.46
4.1.1 Detection of angiotensin-II-generating enzyme activities p.46 4.1.2 Correlation between enzyme activity and MES-signal intensity p.48 4.1.3 Comparison of the detection sensitivity of ACE using the MES-or
fluorometric enzyme assay
p.50
4.1.4 Measurement of angiotensin-II-generating activity of renal tissue in presence of protease inhibitors
p.50
4.2 Part II Purification of two angiotensin-II-generating enzymes from porcine renal tissue protein extract
p.51
4.2.1 PPS experiments for the determination of parameters for anion exchange chromatography
p.51
4.2.2 Anion exchange chromatography of porcine renal tissue protein extract p.53 4.2.3 Inhibitor profiling of the eluates of the anion exchange chromatography
with angiotensin-II- generating activities
p.53
4.2.4 PPS experiments for the determination of parameters for the intermediate chromatographic purification step
p.54
4.2.5 Hydroxyapatite chromatography p.55
4.2.6 PPS experiments for the determination of parameters of lentil lectin affinity chromatography
p.56
4.2.7 Lentil Lectin affinity chromatography p.57 4.2.8 Inhibitor profiling of the angiotensin-II-generating activities of eluates from
lectin affinity chromatography
p.58
4.2.9 Chymostatin-Antipain affinity chromatography p.59
4.2.10 Size exclusion chromatography p.60
4.3 Part III- Purification of an angiotensin-II-generating enzymes from porcine renal tissue protein extract
p.62
4.3.1 Anion exchange chromatography of porcine renal tissue protein extract p.62
4.3.2 Wheat germ lectin chromatography p.62
4.3.3 Inhibitor profiling experiment of the eluate with angiotensin-II-generating activities
p.63
4.3.5 Determination of the molecular weight of the Fraction III p.65 4.3.6 Test of substrate specifity of Fraction III p.67 4.3.7 Database matching of the retrieved substrate specifity information of
Fraction III
p.70
4.4 Part IV- Protein identification of the Fraction I,II and III p.71 4.4.1 Separation of proteins by gel electrophoresis p.71 4.4.2 In- gel digestion of protein bands with specific endopeptidases p.72
4.4.3 Peptide mass fingerprint p.72
4.4.4 NanoLC-ESI-MS/MS of the proteolytic peptides of Fraction II p.73 4.4.5 NanoLC-ESI-MS/MS of the proteolytic peptides of Fraction III p.76
5. Discussion p.77
6.1 Summary p.88
6.2 Zusammenfassung p.89
7. Literature p.90
8. List of figures and tables p.97
9. List of abbreviations p.101
10. Acknowledgement p.104
11. Curriculum vitae p.105
Curriculum vitae page 105 Curriculum vitae
Persönliches
Name: Jana Rykl
Geburtstag: 25. Januar 1978 Geburtsort: Strausberg Nationalität: Deutsch
Ausbildung:
1997 Abschluss der Oberschule mit Abitur
04/2002 Abschluss des Studium der Biotechnologie an der Technischen Fachhochschule Berlin als Diplom-Ingenieurin für Biotechnologie
06/2002-01/2005 Doktorand an der Charité Berlin Campus Benjamin Franklin
Arbeitplätze
06/1997-12/1997 Vorpraktikum am Institut für Molekulare Pharmakologie in Berlin, Abteilung für NMR unterstützte Strukturforschung,
03/2002-09/2000 Praktisches Studiensemester als Stipendiat der Carl-Duisberg-Gesellschaft am Kihara-Institut der Universität Yokohama, Abteilung „Plant Protein Engineering“, Japan
09/2001-03/2002 Diplomsemester als Stipendiat des Deutschen Akademischen Austauschdienstes am Kihara-Institut der Universität Yokohama, Abteilung „Cell Biology“, Japan
06/2002-01/2005 Wissenschaftlicher Mitarbeiter am Institut für Toxikologie der Charité Berlin, Abteilung Endokrinologie und Nephrologie
ab 04/2006 Wissenschaftlicher Mitarbeiter der Firma Cellgenix in der Abteilung „Molecular Therapeutics“ in Freiburg
